Fig. 5: The BAK latch domain induces LC3 lipidation.
From: An unconventional autophagic pathway that inhibits ATP secretion during apoptotic cell death
a BAK scheme showing relevant amino acid positions for the constructs used in (b). b HEK-293T cells were transfected with AU-BAK-ΔBH3 constructs (Nt: M1-M71; Nt-core-ΔBH3: M1-G146 lacking G72-R88 (BH3); latch: G146-G186; all including the transmembrane (TM) domain: P187-S211) and HA-LC3, and lysed 36 h later for Western blot. Relative densitometric quantifications of LC3-II are shown (bottom). c HeLa cells were transfected with the indicated BAK constructs and GFP-LC3, and processed for microscopy 36 h later. Shown are representative confocal pictures (left; green: GFP-LC3, blue: DAPI; scale bars represent 10 μm), and quantification of the number of GFP-LC3-positive puncta per cell (right). Graph bars indicate control vector (white), BAK-ΔBH3 (light grey) and BAK-latch domain (dark grey). Data are displayed as box-plots where the central line represents the median value, the box shows percentiles 25-75 and the whiskers include the most extreme values (n = 25 cells; n.s. P > 0.05, two-tailed Student’s t-test). The numeric P-value is shown. d Alignment of the BAK and BCLXL latch domains showing functionally conserved (red font) and identical (red boxes) amino acids. Positions of the BH2 domains and α6 and α7 helices of BAK are indicated. e HEK-293T cells were transfected with the shown GFP-BAK/BCLXL latch chimeric constructs (right) and HA-LC3, and lysed 36 h later for Western blotting (left). f Scheme showing wild-type and 5M BAK latch domain versions. Boxed positions in the 5M version indicate BCLXL residues (ND-LEP) that substitute those present in BAK (LH-CIA). g HEK-293T cells were transfected with WT and 5M GFP-BAK-latch constructs and HA-LC3, and lysed 36 h later for Western blot. h HeLa cells were transfected with WT and 5M GST-BAK latch versions and GFP-LC3, and processed for microscopy 36 h later. Shown are representative confocal pictures (left; green: GFP-LC3, blue: DAPI; scale bars represent 10 μm), and quantification of the number of GFP-LC3-positive puncta per cell (right). Graph bars indicate control vector (white), BAK-latch-WT (light grey) and BAK-latch-5M (dark grey). Data displayed as in (c) (n = 25 cells; ****P < 0.0001 two-tailed Student’s t-test). Source data are provided as a Source Data file.
